Project Summary The primary purpose of this study is to investigate mechanisms by which commensal gut microbes (gut microbiome) might modulate interindividual differences in tacrolimus metabolism. Tacrolimus is commonly used for immunosuppression after renal transplantation. Its dosing requirements vary widely from person to person, and have proved difficult to predict. This unpredictability has significant clinical implications due to tacrolimus' narrow therapeutic index: subtherapeutic levels increase risk of allograft rejection and supratherapeutic levels increase risk of drug toxicity. Thus, understanding the mechanisms for interindividual differences in tacrolimus metabolism could significantly reduce post-transplant morbidity and mortality. We hypothesize that the gut microbiome influences tacrolimus pharmacokinetics, and might account for interindividual differences in tacrolimus metabolism not explained by existing models. We propose to test this hypothesis by accomplishing two independent and complementary specific aims: I. Identify gut microbial taxa and functions correlating with differences in tacrolimus metabolism through longitudinal surveillance of renal allograft recipients post-transplant. II. Experimentally validate genetic and molecular mechanisms for microbial metabolism of tacrolimus in vitro using functional metagenomic assays, culture, and cloning techniques. In our first Aim, we will follow renal allograft transplants from their pre-transplant evaluation up to one year post-transplant, collecting clinical data, dietary information, and stool samples at monthly intervals. We will use whole-metagenome sequencing to interrogate fecal microbiome taxa and functions at each time point, and identify microbial genes statistically associated with variation in tacrolimus pharmacokinetics. This Aim is innovative because, to our knowledge, no prior studies of the gut microbiome and tacrolimus metabolism have included community encoded microbial functions and host dietary patterns in their analysis. Integrating microbiome functions with clinical and dietary data will ensure the robustness and relevance of our results. In our second Aim, we will functionally screen a large set of previously constructed fecal metagenomic libraries to select for genes involved in tacrolimus metabolism or tolerance. This Aim is innovative because, although direct metabolism of tacrolimus by bacteria has been documented, the molecular mechanisms of microbial tacrolimus metabolism remain unknown. The molecular mechanisms found in our functional metagenomic assays will inform the results of the longitudinal study in Aim 1, and keep them grounded in physiology. Neither Aim depends on the success of the other, but they will work synergistically to provide insight into the molecular mechanisms by which gut bacteria might mediate interindividual differences in tacrolimus metabolism. Understanding the physiology of gut microbiome contributions to tacrolimus pharmacokinetics could lead to future advances that improve the safety and efficacy of tacrolimus therapy.